SlpA as a tool for recombinant protein and enzyme technology

ABSTRACT

Disclosed are a recombinant DNA molecule encoding a fusion protein comprising a SlpA chaperone and a target polypeptide wherein human FK506 binding proteins (FKBPs) are excluded as target polypeptides, a corresponding expression vector encoding said fusion protein as well as host cells transformed with said expression vector. Also disclosed are a method for producing the fusion protein, a recombinantly produced fusion protein comprising a SlpA chaperone and a target polypeptide. A further aspect of the invention is the use of the recombinantly produced fusion protein, and a reagent kit containing a recombinantly produced fusion protein comprising a SlpA chaperone and a target polypeptide.

RELATED APPLICATIONS

This application claims priority to European patent application EP08009537.5 filed May 26, 2008.

FIELD OF THE INVENTION

The present invention relates to fusion proteins comprising a SlpAchaperone and a target polypeptide, methods of recombinantly expressing,purifying and refolding these fusion proteins, their uses in protein andenzyme biotechnology, and particularly their applications indiagnostics. Further, the invention relates to any complex comprisingSlpA and a target polypeptide, which is intended to increase thesolubility, the activity, the stability and/or the folding reversibilityof the target polypeptide or enzyme for biotechnological applications.

BACKGROUND OF THE INVENTION

Protein folding is a spontaneous process that is driven by the smalldifference in Gibbs free energy between the native and unfolded state.Within the folding process, a largely unstructured polypeptide chainadopts what is termed the native conformation or three-dimensionalstructure of a protein. Aggregation of incompletely folded moleculescompetes with productive folding, and this constitutes a major problemand affects the folding yields both in vivo and in vitro. In livingcells, folding is assisted by helper proteins. Folding helpers arepolypeptides that assist the folding and maintain the structuralintegrity of other proteins. They possess the ability to promote theproper folding of a polypeptide chain by reversibly interacting withtheir target, thereby preventing detrimental side reactions such asaggregation processes. They do so both in vivo and in vitro, and thereis an ever increasing number of applications of these folding helpers inbiotechnological problems. Generally, folding helpers are subdividedinto folding catalysts and chaperones.

Chaperones are known to reversibly bind to denatured, partiallydenatured or, put simply, hydrophobic surfaces of polypeptides and thushelp to renature proteins or to keep them in solution. Chaperones lowerthe concentration of aggregation-prone folding intermediates andaggregation-prone folded proteins by reversibly binding and maskinghydrophobic surfaces. They thus exert a mere binding function. Incontrast, folding catalysts such as disulfide oxidoreductases andpeptidyl-prolyl cis/trans isomerases accelerate rate limiting steps inprotein folding and thus shorten the lifetime of folding intermediates.Folding catalysts thus lower the concentration of aggregation-pronefolding intermediates due to their catalytic function. An importantclass of folding catalysts is referred to as peptidyl prolyl cis/transisomerases (PPIases).

Based on sequence similarity, protein topology and binding ofimmnunosuppressant molecules, prolyl isomerases are distinguished intothree distinct families, the cyclophilins, the parvulins and the FK506binding proteins (hence the acronym FKBPs). FKBPs bind to and areinhibited by FK506, rapamycin and related macrolide derivatives, whichhave been used as immunosuppressant drugs.

A putative folding helper that belongs to the FKBP family of peptidylprolyl cis/trans isomerases in E. coli is SlpA, SlpA being the acronymfor “SlyD-like protein A” (Hottenrott et al. 1997, JBC 272/25,15697-15701). Up to now, information on SlpA and its physiological rolein E. coli has been scarce. Although a poor prolyl isomerase activity ofSlpA has been reported, this protein has hitherto remained ratherenigmatic. So far, information on the physico-chemical or possiblechaperone properties of SlpA has been lacking, and the function of SlpAin the E. coli cytosol has not even been addressed.

In many diagnostic applications recombinantly produced proteins are usedas binding partners, e.g., as antigens in an immunoassay designed forthe detection of a specific immunoglobulin analyte. These antigens maybe produced as fusion proteins containing one part that makes up thetarget portion or antigenic polypeptide which is intended to recognizeand bind a specific moiety present in the sample or in the assay mixtureunder study. The other part of the recombinantly produced fusion proteinis a polypeptide portion that is fused to the specificity-conferringantigenic part in order to facilitate its cloning, expression,overproduction, folding/refolding and purification, and to increase itssolubility, its stability or its reversibility of folding. The synthesisof recombinantly produced fusion proteins is well described in priorart. It is also well-established that it is advantageous to usechaperones as that part of the fusion protein that serves a role as ahelping molecule for the expression, folding, purification,solubilization, and the increase in the overall stability of the targetpolypeptide.

U.S. Pat. No. 6,207,420 discloses a fusion protein system for theexpression of proteins, in which the amino acid sequences of the targetpolypeptide part and the fused peptide part originate from differentorganisms. Recently it could be shown that FkpA and SlyD are suitable asfusion modules for the production of recombinant proteins. Bothchaperones increase the expression rate of their client proteins in aprokaryotic host, support correct refolding and increase the overallsolubility of even extremely aggregation-prone proteins such asretroviral transmembrane proteins (Scholz et al. 2005, JMB 345,1229-1241 and WO 03/000877).

While FkpA and SlyD are particularly useful in helping difficult oraggregation-prone proteins to adopt and maintain their native structurein diagnostic reagents and, more generally speaking, biotechnologicalapplications, there remains the challenge of thermal stability. Thenative conformation of proteins is stabilized by a carefully balancednetwork of van-der-Waals contacts, hydrogen bonds, salt bridges andhydrophobic interactions. These contacts are optimized for themicroenvironment of the respective protein, and changes in pH, ionicstrength or temperature do perturb and shift the equilibrium betweenfolded and unfolded molecules. An increase in temperature isparticularly well suited to denature proteins, which often results inaggregation of the fully or partially unfolded molecules. Thermallyinduced aggregation of proteins with the concomitant loss of functionconstitutes a major problem of any protein formulation. It is wellconceivable that elevated temperatures, or, more generally speaking,thermal stress may occur during inappropriate shipment or storage ofprotein reagents or formulations.

A chaperone fusion module such as SlyD, for instance, shows an onset ofthermally induced unfolding at a temperature around 42° C., atemperature which is easily exceeded, e.g., when the cooling system isdefective in a container used for transportation, shipment or storage ofa protein formulation. In case the target protein X is highlyhydrophobic and fully depends on the chaperoning activity of its fusionpartner, the complete fusion polypeptide will aggregate as soon as theSlyD module unfolds and concomitantly loses its solubilizing function.In other words, the stability of SlyD limits the overall stability of aSlyD-X fusion polypeptide when X is a very hydrophobic andaggregation-prone client protein.

Fusion proteins comprising FkpA show a slightly increased stability,probably due to the higher intrinsic thermostability of the dimeric FkpAcarrier module. The melting temperature of E. coli SlyD has beendetermined at around 42° C., whereas FkpA is rather stable up to around50° C. Yet, for reasons that are outlined in the following section,there remains the urgent need to provide alternative functionalchaperone variants with high intrinsic stability.

In a heterogeneous immunoassay of the double antigen sandwich (DAGS)format, for instance, two variants of an antigen are employed on eitherside of the assay. One of these variants bears a label with a highaffinity for the solid phase, the other bears a signaling moiety inorder to generate a signal output. Each of these antigen variants may befused to a helper sequence, i.e., a carrier or fusion module. At leastone chaperone (or a functional polypeptide binding domain, i.e. achaperone domain) is attached or fused to the target polypeptide andfacilitates folding, increases stability and solubility and maintainsthe target polypeptide in a suitable conformation so that the antibodyanalyte to be determined can specifically recognize and bind the targetpolypeptide. Preferably, different chaperones are used as fusionpartners on either side of an immunological bridge assay, in order tobreak the inherent symmetry of the assay. An assay format containingdifferent carrier or fusion modules but identical or similar targetpolypeptides on either side (i.e., on the capture and the detectionside) may also be termed an asymmetric DAGS format. By using differentfusion modules on each side of a DAGS assay, the risk of immunologicalcross-reactions due to the carrier modules and, concomitantly,erroneously high signals may be substantially reduced.

Clearly, the overall stability of the assay is limited by theimmunological component with the lowest inherent stability. When usingFkpA and SlyD as fusion partners in an asymmetric DAGS, SlyD is thefusion partner that limits the overall stability. Thus, there is anobvious need to find other chaperones, which can fully replace SlyDfunctionally and which are inherently more stable towards thermalstress. Even though a wealth of SlyD homologues from thermophilic orhyperthermophilic organisms have been described, there is a caveat insimply using these proteins as fusion partners: Since they have beenevolved and optimized for temperatures far beyond 60° C. they possess anextremely high thermodynamic stability. As a consequence, stable andhyperstable proteins often tend to become rather rigid at ambienttemperature, i.e., they lose the flexibility which is a prerequisite fordynamic binding to target molecules. It is widely accepted that thestability of a protein can only be increased at the expense of both itsflexibility and function, which often precludes highly stable proteinsfrom applications at ambient temperature. An object of the presentinvention is therefore to identify thermostable folding helpers frommesophilic organisms. A further object of the present invention is toprovide polypeptides suitable for diagnostic and biotechnologicalapplications that possess an increased thermal stability and prolong theshelf life of diagnostic reagents and protein formulations.

A few proteins of E. coli are stable and soluble at temperatures farbeyond 49° C. as reported recently by Kwon et al. (BMB reports 2008,41(2), 108-111). The proteins that were soluble upon exposure toelevated temperature were identified by SDS polyacrylamide gelelectrophoresis. The study was carried out with sonicated extracts of E.coli after incubation at various temperatures. Amongst the 17heat-stable proteins that were identified, 6 proteins turned out to beputative folding helpers (GroEL, GroES, DnaK, FkpA, trigger factor,EF-Ts). It is noteworthy that the experiment was performed with acell-free lysate of E. coli and that the solubility of the respectiveprotein was taken as the sole criterion for stability.

There is, however, a substantial difference between the solubility andthe stability of a protein. It is well known in the art that thesolubility of a protein often reaches a minimum at conditions of maximalstability. For instance, the thermodynamic stability of a proteinreaches a maximum when the pH of the buffer solution coincides with thepI of the respective protein. Yet, under these very conditions, theprotein solubility reaches a minimum. Another popular example is thesalting-out of proteins by means of ammonium sulfate or othercosmotropic agents: here also, the solubility of a protein decreases asits stability is increased (ammonium sulfate is a strongly cosmotropicagent, i.e., it stabilizes protein structures).

WO 2007/077008 discloses recombinantly produced chimeric fusion proteinsthat contain the polypeptide binding segment of a non-human chaperonelike, e.g., E. coli SlyD, and N- and C-terminally fused thereto a humanFKBP type peptidyl-prolyl-cis/trans isomerase. A similar fusionpolypeptide has been disclosed using a chaperone segment of SlpA.

Surprisingly, SlpA, in particular E. coli SlpA, is able to conferthermal stability on other target polypeptides when used as a fusionpartner. As reported by Hottenrott et al. (supra) SlpA is an enzyme witha rather poor peptidyl-prolyl cis/trans isomerase activity.Unexpectedly, SlpA exhibits also pronounced chaperone features and, evenmore surprisingly, SlpA possesses an high intrinsic stability andconfers thermal stability on a fused target polypeptide thereby makingthe target polypeptide less susceptible to heat-induced aggregation.Whereas the closely related SlyD exhibits only a marginal stability witha midpoint of thermal unfolding at around 42° C., SlpA retains itsnative fold at least up to 50° C. and shows a midpoint of thermalunfolding (defined as the melting temperature) at around 56° C. This isindeed puzzling given the close relationship between SlyD and SlpA(which stands for SlyD-like protein) and given the fact that both aremonomeric proteins from a mesophilic organism such as E. coli with amaximum growth temperature of 49° C. Most surprisingly, the mesophilicorganism E. coli harbors a putative folding helper such as SlpA thatcombines outstanding thermostability and chaperone features.

SUMMARY OF THE INVENTION

The present invention relates to a recombinant DNA molecule encoding afusion protein comprising a SlpA chaperone and a target polypeptide, acorresponding expression vector encoding said fusion protein as well ashost cells transformed with said expression vector. Another aspect ofthe invention is a method for producing said fusion protein as well as arecombinantly produced fusion protein comprising a SlpA chaperone and atarget polypeptide. A further aspect of the invention is the use of therecombinantly produced fusion protein as a binding partner (such as anantigen, an enzyme or a recombinant calibrator material) or as a meansfor the reduction of interferences in an immunoassay. Further theinvention relates to the use of the recombinantly produced fusionprotein as an immunogen for the production of antibodies against thetarget polypeptide and to the use of the recombinantly produced fusionprotein in the production of a vaccine. Yet another aspect is a methodfor the detection of an analyte in an immunoassay using a recombinantlyproduced fusion protein as well as a reagent kit containing arecombinantly produced fusion protein comprising a SlpA chaperone and atarget polypeptide. A further aspect concerns the use of SlpA as a meansfor the reduction of interferences and cross-reactions in immunoassays.Yet another aspect of the invention is the use of soluble and functionalcomplexes comprising SlpA and a target protein intended forbiotechnological applications, whereby the target protein may be oftherapeutic or diagnostic value.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Near-UV CD spectrum of SlpA from E. coli. The spectrum wasrecorded on a Jasco-720 spectropolarimeter in a thermostated cell holderat 20° C. The protein concentration was 417 μM in a 1 cm cuvette. Thebuffer was 50 mM potassium phosphate pH 7.5, 100 mM KCl, 1 mM EDTA. Bandwidth was 2 nm, resolution was 0.5 nm, the scanning speed was 50 nm/minat a response of 2 s. Spectra were recorded 9 times and averaged inorder to improve the signal-to-noise ratio. The signal was converted tomean residue ellipticity (given in deg cm² dmol⁻¹). The spectrum pointsto a native-like folded protein, the signal maximum is at 262 nm.

FIG. 2: Near-UV CD spectrum of SlyD from E. coli. The spectrum wasrecorded on a Jasco-720 spectropolarimeter in a thermostated cell holderat 20° C. The protein concentration was 200 μM in a 1 cm cuvette. Thebuffer was 50 mM potassium phosphate pH 7.5, 100 mM KCl, 1 mM EDTA. Bandwidth was 2 nm, resolution was 0.5 nm, the scanning speed was 50 nm/minat a response of 1 s. Spectra were recorded 9 times and averaged inorder to improve the signal-to-noise ratio. The signal was converted tomean residue ellipticity (given in deg cm² dmol⁻¹). The spectrum of SlyDsignificantly differs from the spectrum of SlpA. It points to anative-like folded protein, the signal maximum is at 275 nm.

FIG. 3: Thermally induced unfolding transitions of SlyD and SlpA asmonitored by near-UV CD at 275 nm (SlyD) and 262 nm (SlpA). The meltingcurves are normalized to the fraction of native molecules. Unfolding ofboth SlyD and SlpA is fully reversible, and the near-UV CD signal of thenative molecules can be fully restored after the thermal transition whenthe sample is chilled to ambient temperature. The melting temperature(i.e., the temperature at which 50% of the molecules are folded and 50%are unfolded) is 42° C. for SlyD and 56° C. for SlpA. FIG. 3 clearlyillustrates the superior thermal stability of SlpA.

FIG. 4: Near-UV CD spectrum of the SlpA-gp41 fusion protein. Thespectrum was recorded on a Jasco-720 spectropolarimeter in athermostated cell holder at 20° C. The protein concentration was 18.7 μMin a 1 cm cuvette. The buffer was 50 mM potassium phosphate (pH 7.5),100 mM KCl, 1 mM EDTA. Bandwidth was 2.0 nm, resolution was 0.5 nm, thescanning speed was 50 nm/min at a response of 2 s. Spectra were recorded9 times and averaged in order to improve the signal-to-noise ratio. Thesignal was converted to mean residue ellipticity (given in deg cm²dmol⁻¹). The spectrum points to a native-like folded protein. The signalminimum at 293 is indicative of a native-like folded gp41 ectodomainfragment, which is rich in tryptophan residues and absorbs light beyond280 nm. The signature around 290 nm unambiguously points to anative-like fold of the gp41 moiety within the SlpA-gp41 fusionpolypeptide.

FIG. 5: Near-UV CD spectrum of the SlyD-gp41 fusion protein. Thespectrum was recorded on a Jasco-720 spectropolarimeter in athermostated cell holder at 20° C. The protein concentration was 14.4 μMin a 1 cm cuvette. The buffer was 50 mM potassium phosphate (pH 7.5),100 mM KCl, 1 mM EDTA. Bandwidth was 2.0 nm, resolution was 0.5 nm, thescanning speed was 50 nm/min at a response of 2 s. Spectra were recorded9 times and averaged in order to improve the signal-to-noise ratio. Thesignal was converted to mean residue ellipticity (given in deg cm²dmol⁻¹). The signal minimum at 293 is indicative of a native-like foldedgp41 ectodomain fragment, which is rich in tryptophan residues andabsorbs light beyond 280 nm. The signature around 290 nm strongly pointsto a native-like fold of the gp41 moiety within the SlyD-gp41 fusionpolypeptide.

FIG. 6 A/B: The thermally induced unfolding of SlyD-gp41 (A) andSlpA-gp41 (B) is monitored via the decrease in the circular dichroicsignal at 270 nm. Unfolding of the respective chaperone fusion partnergoes along with the loss of its solubilization capacity and leads tospontaneous aggregation of the extremely hydrophobic gp41 moiety. Theonset of aggregation is about 40° C. for SlyD-gp41 and about 56° C. forSlpA-gp41. The ellipticity is given in millidegrees (mdeg), the criticaltemperature boundaries beyond which (irreversible) aggregation occursare highlighted by dashed lines.

FIG. 7: Thermally induced unfolding transition of SlyD-gG1 (26-189) asmonitored by near-UV CD at 280 nm. The ellipticity of the fusion proteinas a function of temperature is given in millidegrees (mdeg). Unfoldingof SlyD-gG1 (26-189) is largely reversible, and the near-UV CD signal ofthe native fusion polypeptide is restored to a large extent when thesample is chilled to ambient temperature. The melting temperature (i.e.,the temperature at which 50% of the molecules are folded and 50% areunfolded) of SlyD-gG1 (26-189) approximates to 53° C.

FIG. 8: Thermally induced unfolding transition of SlpA-gG1 (26-189) asmonitored by far-UV CD at 220 nm. The ellipticity of the fusion proteinas a function of temperature is given in millidegrees (mdeg). Unfoldingof SlpA-gG1 (26-189) is largely reversible, and the far-UV CD signal ofthe native fusion polypeptide is restored to a large extent when thesample is cooled to room temperature. The melting temperature (i.e., thetemperature at which 50% of the molecules are folded and 50% areunfolded) of SlpA-gG1 (26-189) approximates to 63° C. This clearlyillustrates the superior thermal stability of SlpA-gG1 (26-189) whencompared to SlyD-gG1 (26-189).

FIG. 9: Immunological reactivity of SlpA-gG1 (26-189) and SlyD-gG1(26-189) with human anti-HSV-1 positive and anti-HSV-1 negative sera inan automated ELECSYS analyzer (Roche Diagnostics GmbH) as described inExample 4. Table 1 demonstrates the performance of both antigen variantsbefore and after a harsh overnight heat-treatment at 60° C. The outcomeof the experiments clearly shows the superiority of heat-stressedSlpA-gG1 (26-189) over heat-stressed SlyD-gG1 (26-189) in a twofoldmanner. Firstly, the signal recovery with anti-HSV-1 positive sera(upper half of Table 1) is significantly higher with the heat-stressedSlpA fusion polypeptide. Secondly, the increase in background signalwith anti-HSV-1 negative sera (lower half of Table 1) is significantlylower with the heat-stressed SlpA fusion polypeptide. Both effectsimprove the signal dynamics of the immunoassay and highlight theadvantages of SlpA as a stability- and solubility-conferring fusionpartner for difficult target proteins. Thus, the sensitivity of animmunoassay can be warranted by using SlpA as a fusion partner insteadof the closely related SlyD.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 shows the complete amino acid sequence (149 amino acids) ofE. coli SlpA, taken from the SwissProt database accession no. P0AEM0.

MSESVQSNSA VLVHFTLKLD DGTTAESTRN NGKPALFRLG DASLSEGLEQ HLLGLKVGDKTTFSLEPDAA FGVPSPDLIQ YFSRREFMDA GEPEIGAIML FTAMDGSEMP GVIREINGDSITVDFNHPLA GQTVHFDIEV LEIDPALEA

SEQ ID NO: 2 shows the amino acid sequence of E. coli SlpA (amino acidsserine 2 to glutamic acid 148) as used in the Examples section. TheN-terminal methionine is removed cotranslationally in E. coli. Tofacilitate cloning the C-terminal alanine has been removed as well.Further, a C-terminal hexa-histidine tag (SEQ ID NO: 18) has been addedto facilitate purification and refolding of the protein:

SESVQSNSAV LVHFTLKLDD GTTAESTRNN GKPALFRLGD ASLSEGLEQH LLGLKVGDKTTFSLEPDAAF GVPSPDLIQY FSRREFMDAG EPEIGAIMLF TAMDGSEMPG VIREINGDSITVDFNHPLAG QTVHFDIEVL EIDPALEHHH HHH

SEQ ID NO: 3 shows the amino acid sequence of E. coli SlpA-gp41. Thegp41 part contains amino acids 536-681 of HIV 1 gp41, the SlpA partcontains amino acids 1-146. The sequence bears a C-terminalhexa-histidine tag (SEQ ID NO: 18) that has been added to facilitate thepurification and the refolding of the fusion protein.

MSESVQSNSA VLNHFTLKLD DGTTAESTRN NGKPALFRLG DASLSEGLEQ HLLGLKVGDKTTFSLEPDAA FGVPSPDLIQ YFSRREFMDA GEPEIGAIML FTAMDGSEMP GVIREINGDSITVDFNHPLA GQTVHFDIEV LEIDPAGGGS GGGSGGGSGG GSGGGSGGGT LTVQARQLLSGIVQQQNNEL RAIEAQQHLE QLTVWGTKQL QARELAVERY LKDQQLLGIW GCSGKLICTTAVPWNASWSN KSLEQIWNNM TWMEWDREIN NYTSLIHSLI EESQNQQEKN EQELLELDKWASLWNWFNIT NWLWYLEHHH HHH

SEQ ID NO: 4 shows the amino acid sequence of E. coli SlpA-SlpA-gp41.Two SlpA units are attached to the HIV gp41 ectodomain, whichconstitutes a strongly aggregation-prone target polypeptide. The firstSlpA unit comprises amino acids 1-146, the second SlpA unit comprisesamino acids 2-149 (both SlpA variants are fully equivalent in terms offunction and stability). A C-terminal hexa-histidine tag (SEQ ID NO: 18)has been added to facilitate the purification and the refolding of thefusion protein.

MSESVQSNSA VLVHFTLKLD DGTTAESTRN NGKPALFRLG DASLSEGLEQ HLLGLKVGDKTTFSLEPDAA FGVPSPDLIQ YFSRREFMDA GEPEIGAIML FTAMDGSEMP GVIREINGDSITVDFNHPLA GQTVHFDIEV LEIDPAGGGS GGGSGGGSGG GSGGGSGGGS ESVQSNSAVLVHFTLKLDDG TTAESTRNNG KPALFRLGDA SLSEGLEQHL LGLKVGDKTT FSLEPDAAFGVPSPDLIQYF SRREFMDAGE PEIGAIMLFT AMDGSEMPGV IREINGDSIT VDFNHPLAGQTVHFDIEVLE IDPALEAGGG SGGGSGGGSG GGSGGGSGGG TLTVQARQLL SGIVQQQNNELRAIEAQQHL EQLTVWGTKQ LQARELAVER YLKDQQLLGI WGCSGKLICT TAVPWNASWSNKSLEQIWNN MTWMEWDREI NNYTSLIHSL IEESQNQQEK NEQELLELDK WASLWNWFNITNWLWYLEHH HHHH

SEQ ID) NO: 5 shows the amino acid sequence of E. coli SlyD-gp41. AC-terminal hexa-histidine tag (SEQ ID NO: 18) has been added tofacilitate the purification and the in vitro refolding of the protein.

MKVAKDLVVS LAYQVRTEDG VLVDESPVSA PLDYLHGHGS LISGLETALE GHEVGDKFDVAVGANDAYGQ YDENLVQRVP KDVFMGVDEL QVGMRFLAET DQGPVPVEIT AVEDDHVVVDGNHMLAGQNL KFNVEVVAIR EATEEELAHG HVHGAHDHHH DHDHDGGGSG GGSGGGSGGGSGGGSGGGTL TVQARQLLSG IVQQQNNELR AIEAQQHLEQ LTVWGTKQLQ ARELAVERYLKDQQLLGIWG CSGKLICTTA VPWNASWSNK SLEQIWNNMT WMEWDREINN YTSLIHSLIEESQNQQEKNE QELLELDKWA SLWNWFNITN WLWYLEHHHH HH

SEQ ID NO: 6 shows the amino acid sequence of E. coli SlyD-SlyD-gp41.Two SlyD units are fused to the target polypeptide gp41. A C-terminalhexa-histidine tag (SEQ ID NO: 18) has been added to facilitatepurification and in vitro refolding of the protein.

MKVAKDLVVS LAYQVRTEDG VLVDESPVSA PLDYLHGHGS LISGLETALE GHEVGDKFDVAVGANDAYGQ YDENLVQRVP KVFVMGVDEL QVGMRFLAET DQGPVPVEIT AVEDDHVVVDGNHMLAGQNL KFNVEVVAIR EATEEELAHG HVHGAHDHHH DHDHDGGGSG GGSGGGSGGGSGGGSGGGKV AKDLVVSLAY QVRTEDGVLV DESPVSAPLD YLHGHGSLIS GLETALEGHEVGDKFDVAVG ANDAYGQYDE NLVQRVPKDV FMGVDELQVG MRFLAETDQG PVPVEITAVEDDHVVVDGNH MLAGQNLKFN VEVVAIREAT EEELAHGHVH GAHDHHHDHD HDGGGSGGGSGGGSGGGSGG GSGGGTLTVQ ARQLLSGIVQ QQNNELRAIE AQQHLEQLTV WGTKQLQARELAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLE QIWNNMTWME WDREINNYTSLIHSLIEESQ NQQEKNEQEL LELDKWASLW NWFNITNWLW YHGHDHDHDH HHHHH

SEQ ID NO: 7 shows the amino acid sequence of fusion polypeptideSlpA-gG1. One SlpA unit is fused to the target polypeptide gG1,containing amino acids 26-189 of human herpes simplex virus HSV-1antigen gG1 as used in Example 4.

MSESVQSNSA VLVHFTLKLD DGTTAESTRN NGKPALFRLG DASLSEGLEQ HLLGLKVGDKTTFSLEPDAA FGVPSPDLIQ YFSRREFMDA GEPEIGAIML FTAMDGSEMP GVIREINGDSITVDFNHPLA GQTVHFDIEV LEIDPALEGG GSGGGSGGGS GGGSGGGSGG GPTNVSSTTQPQLQTTGRPS HEAPNMTQTG TTDSPTAISL TTPDHTPPMP SIGLEEEEEE EGAGDGEHLEGGDGTRDTLP QSPGPAFPLA EDVEKDKPNR PVVPSPDPNN SPARPETSRP KTPPTIIGPLATRPTTRLTS KGRPLVPTPQ HTPLFSFLTA SPALDLEHHH HHH

SEQ ID NO: 8 shows the amino acid sequence of fusion polypeptideSlyD-gG1. One SlyD unit is fused to the target polypeptide gG1,containing amino acids 26-189 of human herpes simplex virus HSV-1antigen gG1 as used in Example 4.

MKVAKDLVVS LAYQVRTEDG VLVDESPVSA PLDYLHGHGS LISGLETALE GHEVGDKFDVAVGANDAYGQ YDENLVQRVP KDVFMGVDEL QVGMRFLAET DQGPVPVEIT AVEDDHVVVDGNHMLAGQNL KFNVEVVAIR EATEEELAHG HVHGAHDHHH DHDHDGGGSG GGSGGGSGGGSGGGSGGGPT NVSSTTQPQL QTTGRPSHEA PNMTQTGTTD SPTAISLTTP DHTPPMPSIGLEEEEEEEGA GDGEHLEGGD GTRDTLPQSP GPAFPLAEDV EKDKPNRPVV PSPDPNNSPARPETSRPKTP PTIIGPLATR PTTRLTSKGR PLVPTPQHTP LFSFLTASPA LDLEHHHHHH

SEQ ID NO: 9 shows the amino acid sequence of Pasteurella multocida SlyD(full length) according to Swiss Prot ID: Q9CKP2

MKIAKNVVVS IAYQVRTEDG VLVDEAPVNQ PLEYLQGHNN LVIGLENALE GKAVGDKFEVRVKPEEAYGE YNENMVQRVP KDVFQGVDEL VVGMRFIADT DIGPLPVVIT EVAENDVVVDGNHMLAGQEL LFSVEVVATR EATLEEIAHG HIHQEGGCCG GHHHDSDEEG HGCGCGSHHHHEHEHHAHDG CCGNGGCKH

SEQ ID NO: 10 shows the amino acid sequence of the C-terminallytruncated, cysteine-free Pasteurella multocida SlyD variant that ispreferably used as a chaperone unit in a fusion protein for use in adouble antigen sandwich immunoassay (PmS SlyD 1-156).

MKIAKNVVVS IAYQVRTEDG VLVDEAPVNQ PLEYLQGHNN LVIGLENALE GKAVGDKFEVRVKPEEAYGE YNENMVQRVP KDVFQGVDEL VVGMRFIADT DIGPLPVVIT EVAENDVVVDGNHMLAGQEL LFSVEVVATR EATLEEIAHG HIHQEG

SEQ ID NO: 11 shows the amino acid sequence of E. coli FkpA(full-length) according to Swiss Prot ID P45523.

MKSLFKVTLL ATTMAVALHA PITFAAEAAK PATAADSKAA FKNDDQKSAY ALGASLGRYMENSLKEQEKL GIKLDKDQLI AGVQDAFADK SKLSDQEIEQ TLQAFEARVK SSAQAKMEKDAADNEAKGKE YREKFAKEKG VKTSSTGLVY QVVEAGKGEA PKDSDTVVVN YKGTLIDGKEFDNSYTRGEP LSFRLDGVIP GWTEGLKNIK KGGKIKLVIP PELAYGKAGV PGIPPNSTLVFDVELLDVKP APKADAKPEA DAKAADSAKK

SEQ ID NO: 12 shows the amino acid sequence part of E. coli FkpA that ispreferably used as a chaperone unit in a fusion protein for use in adouble antigen sandwich immunoassay. The sequence is lacking theN-terminal signal sequence (amino acid residues 1-25) and essentiallycorresponds to the mature FkpA (FkpA 26-270)

AEAAKPATAA DSKAAFKNDD QKSAYALGAS LGRYMENSLK EQEKLGIKLD KDQLIAGVQDAFADKSKLSD QEIEQTLQAF EARVKSSAQA KMEKDAADNE AKGKEYREKF AKEKGVKTSSTGLVYQVVEA GKGEAPKDSD TVVVNYKGTL IDGKEFDNSY TRGEPLSFRL DGVIPGWTEGLKNIKKGGKI KLVIPPELAY GKAGVPGIPP NSTLVFDVEL LDVKPAPKAD AKPEADAKAA DSAKK

SEQ ID NO: 13 shows the amino acid sequence of Epstein-Barr Virusnuclear antigen 1 (EBV nuclear antigen 1 or EBNA-1) from position401-641, (EBV=HHV-4=human herpes virus 4); strain B95-8. The completeamino acid sequence of EBNA-1 consists of 641 residues and is accessibleunder Swiss Prot ID P03211. The naturally occurring cysteine residuesare dispensable for the antigenicity of EBNA-1 and have been changed toalanine (underlined) in order to simplify the purification process andto increase the yield of native-like folded soluble protein.

GRRPFFHPVG EADYFEYHQE GGPDGEPDVP PGAIEQGPAD DPGEGPSTGP RGQGDGGRRKKGGWFGKHRG QGGSNPKFEN IAEGLRALLA RSHVERTTDE GTWVAGVFVY GGSKTSLYNLRRGTALAIPQ ARLTPLSRLP FGMAPGPGPQ PGPLRESIVA YFMVFLQTHI FAEVLKDAIKDLVMTKPAPT ANIRVTVASF DDGVDLPPWF PPMVEGAAAE GDDGDDGDEG GDGDEGEEGQ E

SEQ ID NO: 14 shows the amino acid sequence of Epstein-Barr Virusprotein p18, amino acids 1 to 176 (open reading frame BFRF3,HHV-4/B95-8), according to Swiss Prot ID P14348. The naturally occurringcysteine residue at amino acid position 56 is dispensable for theantigenicity of EBV p18 and has been changed to alanine (underlined) inorder to simplify the purification process and to increase the yield ofnative-like folded soluble protein.

MARRLPKPTL QGRLEADFPD SPLLPKFQEL NQNNLPNDVF REAQRSYLVF LTSQFAYEEYVQRTFGVPRR QRAIDKRQRA SVAGAGAHAH LGGSSATPVQ QAQAAASAGT GALASSAPSTAVAQSATPSV SSSISSLRAA TSGATAAASA AAAVDTGSGG GGQPHDTAPR GARKKQ

SEQ ID NO: 15 shows the amino acid sequence of the C-terminal part ofEpstein-Barr Virus protein p18, amino acids 105 to 176 (open readingframe BFRF3, HHV4/B95-8), according to Swiss Prot ID P14348.

AASAGTGALA SSAPSTAVAQ SATPSVSSSI SSLRAATSGA TAAASAAAAV DTGSGGGGQPHDTAPRGARK KQ

SEQ ID NO: 16 shows the amino acid sequence of Epstein-Barr Virusprotein p23, amino acids 1 to 162 (open reading frame BLRF2,HHV-4/B95-8), according to Swiss Prot ID P03197. The naturally occurringcysteine residue at amino acid position 46 is dispensable for theantigenicity of EBV p23 and has been changed to alanine (underlined) inorder to simplify the purification process and to increase the yield ofnative-like folded soluble protein.

MSAPRKVRLP SVKAVDMSME DMAARLARLE SENKALKQQV LRGGAAASST SVPSAPVPPPEPLTARQREV MITQATGRLA SQAMKKIEDK VRKSVDGVTT RNEMENILQN LTLRIQVSMLGAKGQPSPGE GTRPRESNDP NATRRARSRS RGREAKKVQI SD

SEQ ID NO: 17 shows the glycine-rich linker peptide sequenceL=(GGGS)₅GGG as used and shown in example 1 for cloning of theexpression cassettes comprising SlpA and a target polypeptide.

GGGSGGGSGG GSGGGSGGGS GGG

DETAILED DESCRIPTION OF THE INVENTION

An aspect of the current invention is a recombinant DNA molecule,encoding a fusion protein, comprising operably linked at least onenucleotide sequence coding for a target polypeptide and upstream ordownstream thereto at least one nucleotide sequence coding for a SlpAchaperone unit.

The term “recombinant DNA molecule” refers to a DNA molecule which ismade by the combination of two otherwise separated segments of sequenceaccomplished by the artificial manipulation of isolated segments ofpolynucleotides by genetic engineering techniques or by chemicalsynthesis. In doing so one may join together polynucleotide segments ofdesired functions to generate a desired combination of functions.

Polynucleotide sequences are operably linked when they are placed into afunctional relationship with each other. For instance, a promoter isoperably linked to a coding sequence if the promoter controlstranscription or expression of the coding sequence. Generally, operablylinked means that the linked sequences are contiguous and, wherenecessary to join two protein coding regions, both contiguous and inreading frame. However, it is well known that certain genetic elements,such as enhancers, may be operably linked even at a distance, i.e., evenif not contiguous.

The terms “upstream” and “downstream” are functionally defined and referto the direction or polarity of an encoding nucleotide sequence strand.“Upstream” direction means that the nucleotide is located in 5′direction of a given polynucleotide sequence, i.e., towards the startingnucleotide. In terms of amino acid sequence the term “upstream”translates into/means an amino acid that is located in N-terminaldirection, i.e., towards the start of the polypeptide chain. Preferably,the nucleotide sequence encoding a SlpA chaperone unit is locatedupstream of the nucleotide sequence encoding the target polypeptide.

“Downstream” direction means that the nucleotide is located in 3′direction of the polynucleotide, i.e., towards the end of the nucleotidesequence. In terms of amino acid sequence the term “downstream”translated into an amino acid that is located in C-terminal direction,i.e., towards the end of the polypeptide chain.

A polynucleotide is said to “code for” or to “encode” a polypeptide if,in its native state or when manipulated by methods known in the art, thepolynucleotide can be transcribed into a nucleotide template and/or betranslated to yield the polypeptide or a fragment thereof.

Another aspect of the invention is an expression vector comprisingoperably linked a recombinant DNA molecule comprising at least onenucleotide sequence coding for a target polypeptide and upstream ordownstream thereto at least one nucleotide sequence coding for a SlpAchaperone.

DNA constructs prepared for introduction into a host typically comprisea replication system recognized by the host, including the intended DNAfragment encoding the desired target fusion peptide, and will preferablyalso include transcription and translational initiation regulatorysequences operably linked to the polypeptide encoding segment.Expression systems (expression vectors) may include, for example, anorigin of replication or autonomously replicating sequence (ARS) andexpression control sequences, a promoter, an enhancer and necessaryprocessing information sites, such as ribosome-binding sites, RNA splicesites, polyadenylation sites, transcriptional terminator sequences, andmRNA stabilizing sequences.

The appropriate promoter and other necessary vector sequences areselected so as to be functional in the host. Many useful vectors forexpression in bacteria, yeast, mammalian, insect, plant or other cellsare known in the art and are commercially available. In addition, theconstruct may be joined to an amplifiable gene so that multiple copiesof the gene may be obtained.

Expression and cloning vectors will likely contain a selectable marker,a gene encoding a protein necessary for the survival or growth of a hostcell transformed with the vector, although such a marker gene may becarried on another polynucleotide sequence co-introduced into the hostcell. Only those host cells expressing the marker gene will surviveand/or grow under selective conditions. Typical selection genes includebut are not limited to those encoding proteins that (a) conferresistance to antibiotics or other toxic substances, e.g., ampicillin,tetracycline, etc.; (b) complement auxotrophic deficiencies; or (c)supply critical nutrients not available from complex media. The choiceof the proper selectable marker will depend on the host cell, andappropriate markers for different hosts are known in the art.

The vectors containing the polynucleotides of interest can be introducedinto the host cell by any method known in the art. These methods varydepending on the type of the respective host system, including but notlimited to transfection employing calcium chloride, rubidium chloride,calcium phosphate, DEAE-dextran, other substances, and infection byviruses. Large quantities of the polynucleotides and polypeptides of thepresent invention may be prepared by expressing the polynucleotides ofthe present invention in vectors or other expression vehicles incompatible host cells. The most commonly used prokaryotic hosts arestrains of Escherichia coli, although other prokaryotes, such asBacillus subtilis may also be used.

Expression in Escherichia coli represents a preferred mode of carryingout the present invention. Expression of fusion proteins comprising atleast one SlpA unit and at least one target polypeptide X unit orcoexpression of SlpA and X to yield soluble SlpA-X complexes, whetherSlpA and X be covalently linked or not, is feasible in prokaryotic aswell as in eukaryotic host cells.

Construction of a vector according to the present invention employsconventional ligation techniques. Isolated plasmids or DNA fragments arecleaved, tailored, and relegated in the form desired to generate theplasmids required. If desired, analysis to confirm correct sequences inthe constructed plasmids is performed in a known fashion. Suitablemethods for constructing expression vectors, preparing in vitrotranscripts, introducing DNA into host cells, and performing analysesfor assessing expression and function are known to those skilled in theart. Gene presence, amplification and/or expression may be measured in asample directly, for example, by conventional Southern blotting,Northern blotting to quantitate the transcription of mRNA, dot blotting(DNA or RNA analysis), or in situ hybridization, using an appropriatelylabeled probe which may be based on a sequence provided herein. Thoseskilled in the art will readily envisage how these methods may bemodified, if desired.

A further embodiment of the invention is a host cell transformed with anexpression vector comprising operably linked a recombinant DNA moleculecomprising at least one nucleotide sequence coding for a targetpolypeptide and upstream or downstream thereto at least one nucleotidesequence coding for a SlpA chaperone.

Another embodiment of the invention refers to a method of coexpressionof SlpA and a target polypeptide in a prokaryotic or eukaryotic host,whereby the overproduced SlpA interacts with the target polypeptide andforms a soluble non-covalent complex, which facilitates the preparationof native-like folded and active target polypeptide. This means that theDNA sequences encoding SlpA and the target polypeptide may be located onthe same vector and controlled by either identical or differentpromoters. Alternatively, the DNA molecules encoding SlpA and the targetpolypeptide may be located on different compatible vectors. Forsimultaneous expression of SlpA and the target polypeptide host cellsare transformed with both vectors. Preferably, the genes encoding thetarget protein and SlpA are controlled by different promoters, which areresponsive to different inducers. Thus, induction of SlpA and targetprotein may be carried out simultaneously or consecutively in acontrolled and defined manner. For instance, SlpA expression may beinduced first to generate a basal level of functional chaperone, andsubsequently the induction of the target gene may be carried out. Thissequential approach aside, simultaneous induction of folding helper andtarget polypeptide is feasible and may yield soluble and functionaltarget protein as well. The genes encoding SlpA and the target proteinmay be located on the same or on different vectors.

The term “fusion protein” means that two otherwise separatedpolypeptides are functionally combined on a single polypeptide chain.The single elements of the fusion protein, i.e., the SlpA chaperone partand the target polypeptide part, also termed target polypeptide X, maybe directly adjacent to each other. Optionally they are separated by apeptide linker of 1-100 amino acid residues, preferably 5-30 amino acidresidues most preferably around 20 amino acid residues. As the skilledartisan will appreciate such a linker polypeptide is designed as mostappropriate for the intended application, especially with regard tolength, flexibility, charge and hydrophilicity. The linker polypeptidesequence may also contain a proteolytic cleavage site. Optionally thefusion protein may also contain a signal peptide sequence for targetingthe protein to the desired compartment in which folding should takeplace.

According to the invention more than one target polypeptide X, e.g.,two, three or four copies of the target polypeptide may be part of thefusion protein. As an example, SlpA-X2 means that one SlpA unit is fusedto two target polypeptide units of the X type. The single targetpolypeptide units may or may not be separated by a linker polypeptidesegment. The fusion protein contains at least one SlpA chaperone unit.As well, tandem, triple or higher combinations may constitute the fusionprotein, e.g., SlpA-SlpA-X or SlpA-SlpA-SlpA-X. As well, fusion proteinsin which the target polypeptide is sandwiched between at least twochaperone units, are part of the invention, e.g., SlpA-X-SlpA orSlpA-SlpA-X-SlpA-SlpA.

SlpA is a putative peptidyl prolyl cis/trans isomerase of the FKBPfamily. The E. coli SlpA amino acid sequence as published underSwissProt accession no. P0AEM0 is shown in SEQ ID NO: 1.

According to the invention the term “nucleotide sequence coding for aSlpA chaperone” refers to a nucleotide sequence encoding a polypeptidefragment comprising the polypeptide binding segment of SlpA. The term“polypeptide binding segment” of a chaperone denotes thebinding-competent part of the chaperone, i.e., the part that binds andholds the client or substrate polypeptide chain and thus sequesters itto decrease the concentration of aggregation-prone folding intermediatesand to facilitate subsequent folding. The “polypeptide binding segment”of SlpA may also be named IF domain (insert in flap domain). Defined asan autonomous folding unit, a protein domain is able to adopt anative-like stable fold in aqueous solution under appropriate refoldingconditions. The terms “polypeptide binding segment”, “IF-loop”,IF-domain or chaperone domain may be used synonymously.

“SlpA” or “SlpA chaperone” or “SlpA unit” according to the inventioncomprises the polypeptide binding segment or IF domain of SlpA.Preferably, the entire molecule of E. coli SlpA is used as a fusionpartner. Alternatively, the SlpA IF domain may be used as a fusionpartner. It comprises at least a fragment N-terminally starting with anyamino acid located between amino acid no. 59 and 78 of SEQ ID NO: 2 andC-terminally ending with any amino acid located between amino acid no125 and 139 of SEQ ID NO: 2. Most preferred is a sequence coding for apolypeptide N-terminally starting with amino acid no. 72 (Valine 72) andC-terminally ending with amino acid no. 132 (Threonine 132) of SEQ IDNO: 2. According to the invention, SlpA refers to the maturenon-humanized form of this chaperone. This means that the SlpA chaperonedoes neither contain N- nor C-terminally flanking sequences of FKBP12 orany other human FKBP.

According to the invention, SlpA chaperone homologues from otherorganisms may be used as folding helpers combining a prolyl isomerasewith a chaperone activity. Such SlpA homologues may originate from thefollowing organisms (Swiss Prot database ID numbers are denoted inbrackets): Shigella flexneri (Prot.ID. P0AEM3), Shigella sonnei (Prot.IDQ3Z5Y2), Shigella dysenteriae (Prot. ID Q32K69), Citrobacter Koseri(Prot.ID A8ALT4), Salmonella typhi (Prot.IDQ8XG79), Salmonellatyphimurium (Prot.ID Q7CR92), Salmonella paratyphi A and B (Prot.IDQ5PKI5 and A9MYG7), Salmonella cholerasesuis (Prot.ID Q57TL3),Klebsiella pneumoniae (Q9RF46), Salmonella arizonae (Prot.ID A9MR44),Enterobnacter sp. (Prot.ID A4W6E3), Enterobacter sakazakii (A7MIM1),Serratia proteamaculans (Prot.ID A8G9L6), Yersinia pestis (Prot.IDQ8CZP4 or Q0WJI9), Yersinia pseudotuberculosis (Prot.ID A7FMD5),Yersinia enterolitica (A1JJE3), Erwinia carotovora (Prot.ID Q6D0C5),Photorabdus luminescens (Prot.ID Q7N8X0), Sodalis glossinidius (Prot.IDQ2NVY4), Idiomarina ballica (Prot.ID A3WMS1), Vibrio harveyi (Prot.IDA6ATG3 or A7MTD8), Vibrio vulnificus (Prot.ID Q7MNM6 or Q8DES9), Vibriocampbellii (Prot.ID A8T7R0), Vibrio shilonii (Prot.ID A6D8Q3), Vibriosplendidus (Prot.ID A3UXQ8), Idiomarina loihiensis (Prot.ID Q5QZR6),Vibrio alginolyticus (Prot.ID Q1V5T9), Aeromonas salmonicida (Prot.IDA4SIX7), Photobacrerium sp. (Q2C7V1), Vibrio parahaemolyticus (Prot.IDQ87S88 or A6B565), Pseudoalteromonas atlantica (Prot.ID Q15R06), Vibriocholerae (Prot.ID A5F8X4, or Q9KU45, or A6Y5H7, or A6XZU4, or A6ADB4, orA6A5W5, or A3H4C9, or A3GPA9, or A3EG01, or A2PSS5, or A2P8T9, orA1F6Q8), Aeromonas hydrophila (Prot.ID A0KG41), Vibrio angustum (Prot.IDQ1ZMQ4), Moritella sp. (Prot.ID A6FG75), Pseudoalteromonas haloplanktis(Prot.ID Q31EA0), Alteromonadales bacterium (Prot.ID A0Y1B2),Psychromonas ingrahamii (Prot.ID A1SZP1), Vibrio fischeri (Prot.IDQ5E7N2 or A9IPH0), Photobacterium profundum (Prot.ID Q1Z378 or Q6LUK9),Pseudoalteromonas tunicata (Prot.ID A4C627), Psychromonas sp. (Prot.IDQ1ZHS3), Reineka sp. (Prot.ID A41BJL0), Vibrio psychroerythus (Prot.IDQ486T8), Shewanella amazonensis (Prot.ID A1S427), Shewanella sp.(Prot.ID Q0HFZ1, or Q0HS84, or A0KZY9), Shewanella pealeana (Prot.IDA8H1H5), Shewanella frigidimarina (Prot.ID Q07Z37), Shewanelladenitrificans (Prot.ID Q12KM6), Shewanella loihica (Prot.ID A3QBX4), andShewanella purefaciens (Prot.ID A4Y4A6).

According to the invention the SlpA chaperone sequence may be modifiedby amino acid substitutions, preferably homologous substitutions,deletions and insertions provided that the overall structure, functionand stability of the SlpA chaperone is maintained. Maintenance of thefunction of such a SlpA variant may easily be tested by determining themelting temperature of a fusion protein comprising a target polypeptideand the SlpA chaperone sequence under investigation. The meltingtemperature is defined as the temperature at which 50% of the moleculesare folded and 50% are unfolded, i.e., the melting temperaturedetermines the midpoint of the thermally induced unfolding transition ina given buffer system at a given protein concentration. Depending on thecontent in aromatic residues, the melting of proteins can be monitoredby simple spectroscopic probes such as UV absorbance, fluorescence orcircular dichroism. Circular dichroism, in particular, is well-suited tomonitor conformational changes in the secondary structure (amide CD orfar-UV CD) or in the tertiary structure (aromatic CD or near-UV CD) ofproteins.

Thermally induced unfolding of SlpA as assessed by near-UV CD revealsthat the unfolding process is fully reversible, i.e., SlpA spontaneouslyre-adopts its native conformation after the sample is chilled down from95° C. to ambient temperature, i.e., to 15-25° C. This reversibility offolding and unfolding is a pivotal prerequisite for an ideal foldinghelper in biotechnological applications: Often, recombinant fusionproteins accumulate as inclusion bodies in the E. coli cytosol when theyare heavily overproduced. In this case, a robust and efficientrenaturation protocol has to be elaborated, starting off with bacterialcells or inclusion bodies lysed in 7.0 M guanidinium chloride or otherchaotropic agents such as urea. It is self-evident that the refolding ofany chaperone fusion partner must be sufficiently robust, efficient andreversible in order to assist the in vitro refolding of the desiredclient protein. Many fusion partners known in prior art such as, e.g.,NusA, MBP (maltose binding protein) and GST (glutathione-S-transferase),exhibit a very robust de novo folding upon translation in the host cell,but they can not easily be refolded after thermally or chemicallyinduced unfolding. These fusion partners are therefore employed with theaim of soluble expression of the target protein in the host system. Whenthey fail to confer solubility on their client proteins during de novofolding upon translation in the host cell, recovery of the aggregatedfusion proteins by in vitro renaturation attempts is difficult.According to the present invention, a fully reversible fusion partnersuch as SlpA has its obvious advantages in that it may as well lead to asoluble protein production upon de novo folding in the host cell. Inaddition, SlpA, by virtue of its folding reversibility, may as well beused to assist the in vitro refolding of a fusion polypeptide that hasaccumulated in insoluble inclusion bodies upon massive overproduction inthe host cell. Complete reversibility of unfolding in combination with ahigh intrinsic stability and substantial chaperone features areimportant prerequisites of a fusion partner according to the presentinvention. These criteria are perfectly met by SlpA.

According to the invention, one or more, preferably two nucleotidesequences encoding a SlpA chaperone are located upstream of thenucleotide sequence coding for a target polypeptide, resulting in atandem SlpA chaperone comprising two adjacent SlpA units. The one ormore nucleotide sequences encoding a SlpA chaperone may be separated bya nucleotide sequence encoding (in frame) a peptide linker of 1-100amino acids. Different nucleotide sequences may be used to encode thetwo SlpA chaperone units. As well, different nucleotide sequences shouldbe used to encode all the other highly repetitive elements such aslinker or spacer segments within the fusion polypeptide. The nucleotidesequences should be degenerated in order to avoid the loss of one SlpAcoding sequence due to inadvertent recombination events in the E. colihost. By carefully selecting different codons for identical orrepetitive amino acid sequences, the stability of the expressioncassette can be secured.

A “target polypeptide” according to the invention may be any polypeptide(i.e., any amino acid sequence) that is limited in solubility orstability, that tends to aggregate under unfavorable conditions and thatneeds to be supported or assisted by a folding helper with the provisothat FK506 binding proteins (FKBPs), in particular human FK506 bindingproteins, are excluded as target polypeptides. This means that FK506binding proteins such as, e.g., human FKBP12 are excluded as targetpolypeptides. In a preferred embodiment, polypeptides that show atendency to aggregate and/or are susceptible to thermal stress may beused as a target polypeptide. Moreover, polypeptides with enzymaticactivity are preferred target polypeptides according to the invention.In particular, enzymes that accept and turn over hydrophobic substrates(and therefore harbor hydrophobic surface patterns themselves) arepreferred target polypeptides according to the invention. In a furtherpreferred embodiment, bacterial or viral proteins or prion proteins orproteins associated with rheumatoid arthritis are used as targetpolypeptides.

Any structural, membrane-associated, membrane-bound or secreted geneproduct of a mammalian pathogen may be used as a target polypeptide.Mammalian pathogens include viruses, bacteria, single-cell or multi-cellparasites which can infect or inhabit a mammalian host. For example,polypeptides originating from viruses such as human immunodeficiencyvirus (HIV), vaccinia, poliovirus, adenovirus, influenza, hepatitis A,hepatitis B, dengue virus, Japanese B encephalitis, Varicella zoster,cytomegalovirus, Epstein-Barr virus, rotavirus, as well as virusescausing measles, yellow fever, mumps, rabies, herpes, influenza,parainfluenza and the like may be used as a target polypeptide in thefusion protein according to the invention. Bacterial proteins of, e.g.,Vibrio cholerae, Salmonella typhi, Treponema pallidum, Helicobacterpylori, Bordetella pertusis, Streptococcus pneumoniae, Haemophilusinfluenzae, Clostridium tetani, Corynebacterium diphtheriae,Mycobacterium leprae, R. rickettsii, Shigella, Neisseria gonorrhoeae,Neisseria meningitidis, Coccidioides immitis, Borrelia burgdorferi, andthe like may be used as a target polypeptide.

Further examples of target polypeptides preferably produced by thepresent methods include mammalian gene products such as enzymes,cytokines, growth factors, hormones, vaccines, antibodies and the like.More particularly, preferred over expressed gene products of the presentinvention include gene products such as erythropoietin, insulin,somatotropin, growth hormone releasing factor, platelet derived growthfactor, epidermal growth factor, transforming growth factor α,transforming growth factor, epidermal growth factor, fibroblast growthfactor, nerve growth factor, insulin-like growth factor I, insulin-likegrowth factor II, clotting Factor VIII, superoxide dismutase,interferon, y-interferon, interleukin-1, interleukin-2, interleukin-3,interleukin-4, interleukin-5, interleukin-6, granulocyte colonystimulating factor, multi-lineage colony stimulating factor,granulocyte-macrophage stimulating factor, macrophage colony stimulatingfactor, T cell growth factor, lymphotoxin and the like. Preferred overexpressed gene products are human gene products.

For diagnostic purposes, when, e.g., the analyte to be determined is anantibody, the target polypeptides contain at least one epitope that isrecognized by the antibodies to be determined. Such epitopes are alsocalled diagnostically relevant epitopes. A target polypeptide accordingto the invention may also comprise sequences like, e.g., diagnosticallyrelevant epitopes from several different proteins constructed to beexpressed as a single recombinant polypeptide. Preferably, the targetpolypeptide has a length of 10-500 amino acids.

Most preferably, the target polypeptide is a member of a groupconsisting of retroviral proteins such as gp41 and p17 from HIV-1, gp36and p16 from HIV-2, gp21 from HTLV-I/II, consisting of viral envelopeproteins such as E1 and E2 from Rubella virus or consisting ofamyloidogenic proteins such as β-AP42 (Alzheimer peptide) or prionprotein.

Also preferred as target polypeptides are the glycoprotein G1 fromherpes simplex virus 1 and the glycoprotein G2 from herpes simplex virus2. More exactly, the respective glycoprotein fragments lacking theirsignal sequences and their transmembrane regions (gG1 26-189, gG2343-594) are suitable target polypeptides.

Further preferred as target polypeptides are the following proteins andprotein fragments from Human Cytomegalovirus: pp28 (15-179), pp150(821-1048), pp150 (547-725), pp150 (495-854), p38 (105-308), p38(105-373), p38 (209-308), p52 (254-293), p52 (295-330), p52 (298-433),gB (67-84), pp65 (372-549), and pp65 (372458).

Also preferred are the following proteins and protein fragments fromTreponema pallidum: TpN17 (23-156), TpN47 (21-434), TpN15 (23-142), TmpA(23-345), TpO453 (27-287). The signal sequences of all these Treponemaantigens have been omitted to ensure cytosolic localization uponexpression in the E. coli host.

Further preferred target polypeptides are the following proteins andprotein fragments from Borrelia: internal flagellin fragment p41i(137-262), V1sE (IR6/C6), DbpA (26-175), OspB (17-296), and OspC(19-214).

Further preferred target polypeptides are proteins from Epstein-Barrvirus (EBV) such as EBV nuclear antigen 1 (EBNA-1) as shown in SEQ IDNO: 13, polypeptides and fragments of p18 as shown in SEQ ID NOs: 14 and15, respectively and polypeptides derived from p23 as shown in SEQ IDNO: 16.

Any of these target polypeptides when fused to a SlpA chaperone can beused in an immunoassay as a binding partner for the detection of ananalyte like, e.g., antibodies against the target polypeptide or may beused as a standard or calibration material for immnunoassays asdescribed in further detail below.

A further embodiment of the invention is a method of producing a fusionprotein said method comprising the steps of a) culturing host cellscomprising at least one nucleotide sequence coding for a targetpolypeptide and upstream thereto at least one nucleotide sequence codingfor a SlpA chaperone, b) expression of said fusion protein, c)purification of said fusion protein and d) refolding into a soluble andnative-like or immunoreactive (i.e., antigenic) conformation. A fusionprotein produced by this method is also an aspect of the invention.

The fusion proteins according to the invention exhibit high solubility.When over expressed at a low rate in the cytosol they mainly accumulatein the soluble fraction. Depending on the conditions of cell growth andinduction, especially when heavily over expressed, the SlpA-X geneproducts may also accumulate in inclusion bodies. Customarily, theskilled artisan aims at the overproduction of soluble targetpolypeptides in the E. coli cytosol. Cells are then disrupted bysonication or a combined lysozyme/EDTA treatment and the putativelynative-like folded target proteins are isolated from the solublefraction. This is feasible for SlpA-X fusion proteins and leads tosoluble material in case the target polypeptide X possesses asufficiently high intrinsic solubility. In case the target polypeptide Xis very hydrophobic and strongly tends to aggregate, an alternativestrategy may be applied which exploits the efficient and robustrefolding properties of SlpA in a matrix-assisted renaturation approach.Cells are lysed under appropriate buffer conditions like, e.g., inchaotropic substances, which are strongly denaturing and solubilize evenhydrophobic cell components and also the inclusion bodies, albeit at theexpense of structural integrity. When the fusion proteins are N- orC-terminally tagged with a hexa-histidine (SEQ ID NO: 18) moiety, theymay be specifically bound in an unfolded state to a metal-containingcolumn (Ni-NTA or Zn²⁺ or Cu²⁺ supports). Immobilized to the solidphase, the molecules are easily and efficiently refolded underappropriate buffer conditions. This so-called matrix-assistedrenaturation, which has been shown to increase the refolding yield ofmany difficult proteins, is strongly supported by the covalently linkedSlpA, which, by virtue of its chaperone properties, possibly recognizesand reversibly masks hydrophobic patches in folding intermediates.Appropriate purification and refolding protocols as shown in more detailin the Examples section are well known to the skilled artisan.

A further aspect of the invention relates to any complex comprising SlpAand target polypeptide sequence, which includes addition of SlpA to anyprotein formulation. A further aspect of the invention relates to arecombinantly produced fusion protein comprising at least onepolypeptide sequence corresponding to SlpA and at least one polypeptidesequence corresponding to a target polypeptide. A further aspect of theinvention relates to a synthetically produced SlpA either alone or incombination with a target polypeptide of recombinant or syntheticorigin.

According to the invention, a SlpA chaperone is able to improve thethermal stability of difficult target polypeptides when used as a fusionpartner. SlpA confers thermal stability on a fused target polypeptidethereby making the target polypeptide less susceptible to heat-inducedaggregation as shown in the Examples section. When stronglyaggregation-prone target proteins fused to E. coli SlyD are subjected tothermal stress, the resulting fusion proteins show an onset ofthermally-induced aggregation at about 42° C., which is in fairagreement with the inherent stability of SlyD. When the same targetproteins are fused to SlpA, preferably to E. coli SlpA, they remainstable and soluble up to around 56° C. For example, a fusion proteincontaining SlyD and the fragment 536-681 from the HIV protein gp41 (SEQID NO: 5) starts to aggregate at a temperature of 42° C. whereas thesame target protein fused to E. coli SlpA (SEQ ID NO: 3) according tothe invention is thermally stable at temperatures beyond 50° C. It canbe shown that SlpA as part of a fusion protein protects difficult oraggregation-prone proteins against aggregation following heat-induceddenaturation.

It can also be shown that fusion of SlpA exerts a beneficial effect evenon proteins or protein fragments that are less aggregation-prone. Whenthe glycoprotein G1 fragment gG1 (26-189) from HSV-1 is fused to SlyD,the resulting fusion protein can be thermally unfolded in a largelyreversible fashion with an approximate melting temperature at 53° C.(FIG. 7). When, however, the same fragment is fused to SlpA theresulting fusion protein shows a midpoint of thermally induced unfoldingat approximately 63° C. (FIG. 8). Obviously, the stability of the gG1fusion polypeptide is shifted by 10° C. upon substitution of SlpA forSlyD as a fusion partner. This finding clearly demonstrates the superiorstability features of a SlpA-X fusion polypeptide compared to its SlyD-Xcounterpart.

In order to elucidate whether these superior stability features of SlpAfusion polypeptides are also reflected in an immunoassay, thermallychallenged samples of SlyD-gG1 and SlpA-gG1 were assessed with anti-HSVpositive and negative human sera (Example 4) for their immunoreactivityrecovered after heat-stress. When compared with unstressed samples, aclear result was observed (see Example 4 and FIG. 9): The signalgenerated by heat-treated SlyD-gG1 and SlpA-gG1 with anti-HSV positivesera was reduced in all cases, but the signal loss was much morepronounced with the SlyD fusion variant. In turn, the background signalgenerated by heat-treated SlyD-gG1 and SlpA-gG1 with anti-HSV negativesera was increased in all cases (indicative of aggregation processes ofthe ruthenium-conjugated antigen), but the increase in signal height wasagain much more pronounced with the SlyD fusion variant. With respect tothe signal readout for both the positive and the negative sera (i.e.,with respect to the signal dynamics), SlpA is therefore clearly superiorto SlyD as a fusion partner for gG1 (26-189). Obviously, the use of SlpAinstead of SlyD as a fusion partner ensures both a lower signal levelwith negative sera and a higher signal recovery with positive sera.Briefly, the use of SlpA as a fusion partner warrants excellent signaldynamics even after harsh treatment of an immunoassay kit containingpolypeptide antigens for the detection of immunoglobulin analytes. It iswell conceivable that SlpA or related chaperone modules that arecovalently attached to their target molecules via sufficiently long andflexible cross-linkers (by means of standard chemical methods) exert asimilar solubilizing effect. In such cases, where a fusion polypeptideis not feasible, the chaperone and the target molecule would be producedand refolded separately before being linked covalently.

A further aspect of the invention concerns the use of a recombinantly orsynthetically produced fusion protein comprising an SlpA chaperone and atarget polypeptide as a binding partner in an immunoassay. Immunoassaysand various homogenous and heterogeneous test formats are well known tothe skilled artisan. They can be carried out in all biological liquidsknown to someone skilled in the art. Preferred samples are body liquidslike whole blood, blood sera, blood plasma, urine or saliva.

The fusion polypeptides comprising SlpA and at least one targetpolypeptide according to the invention may also be used as standard orcalibration material. SlpA is also a good fusion partner for difficultproteins that are needed as calibrators in immunoassays. For instance,we cloned, expressed and purified a Troponin I variant (comprisingresidues 1-209) in fusion with SlpA. The resulting fusion polypeptideSlpA-Troponin I turned out to be soluble and immunoreactive and waswell-suited as a standard calibrator material for an Troponin Iimmunoassay. Due to the SlpA fusion partner, the stability of theTroponin I moiety was substantially increased when compared to theisolated Troponin I, which is only marginally stable and spontaneouslyaggregates even under favorable buffer conditions. The use of SlpA incomplex with difficult proteins to generate soluble and stablecalibrator or standard materials is a further embodiment of theinvention. Yet a further aspect is the use of SlpA as an additive toimprove the solubility and prevent the aggregation of a target protein.

A further embodiment of the invention is a method for the detection ofantibodies specific for an analyte in an isolated sample, said methodcomprising

-   -   a) forming an immunoreaction admixture by admixing a body fluid        sample with a fusion protein comprising at least one polypeptide        sequence corresponding to a SlpA chaperone and at least one        polypeptide sequence corresponding to a target polypeptide,    -   b) maintaining said immunoreaction admixture for a time period        sufficient for allowing antibodies against said analyte present        in the body fluid sample to immunoreact with said fusion protein        to form an immunoreaction product; and    -   c) detecting the presence of any of said immunoreaction product.

In a preferred embodiment the detection of specific antibodies can beperformed by the so-called double antigen sandwich test (DAGS; alsocalled bridge test), a heterogeneous format wherein the specificantibody analyte to be determined forms a bridge between two identicalor similar antigens. This format can readily be adapted forhigh-throughput automated analyzers. More specifically, the antibodiesto be determined form an immunocomplex or immunoreaction product with afirst antigen which mediates immobilization to a solid phase and with asecond antigen carrying a label (i.e., a signaling moiety like achromogenic, fluorescent, chemiluminescent, electrochemiluminescent orother labels that are known to someone skilled in the art) thus allowingquantitative or qualitative detection of the specifically boundantibodies after separation of the liquid and the solid phase.Therefore, only if the antibodies under investigation are present in thesample a bridge is formed, and a signal can be detected. In such anassay format the fusion proteins according to the invention can be usedas binding partners wherein the solid phase-bound antigen or the labeledantigen or both are fusion proteins comprising an E. coli SlpA chaperoneand a target polypeptide. The target polypeptide constitutes theantigenic part of the fusion protein.

A preferred embodiment of the invention is a so-called asymmetric doubleantigen sandwich test for the detection of a specific antibody wherein afirst fusion protein and a second fusion protein each comprising achaperone and a target polypeptide are used. This format is termedasymmetric because the chaperone units of both fusion proteins differfrom each other. For instance, the first fusion protein may comprise atleast one SlpA chaperone unit and at least one target polypeptide unitand may bear a moiety that mediates specific binding to a solid phaselike, e.g., biotin that binds to a streptavidin-coated solid phase. Thesecond fusion protein may comprise at least one chaperone unit differentfrom SlpA and at least one target polypeptide unit that is identical orsimilar to the target polypeptide of the first fusion protein. Inaddition, the latter fusion protein may carry a signaling moiety or areporter group for signal readout.

Preferably, the chaperone unit of the second fusion protein is also athermostable chaperone with sufficient intrinsic flexibility (i.e.,highly dynamic binding activity) at ambient temperature. Suitablecandidates for the chaperone unit of the second fusion protein are forexample FkpA (melting temperature around 50° C.) and a C-terminallytruncated (cysteine-free) variant of the SlyD orthologue fromPasteurella multocida (melting temperature around 49° C.). The aminoacid sequences of both chaperones (complete sequences and partialsequences preferably used as chaperone unit in a fusion protein) areshown in SEQ ID NOs. 9 to 12. The chaperone unit of the first and secondfusion protein, may be exchanged, i.e., SlpA may be part of the secondfusion protein and in this case the other thermostable chaperone like,e.g., FkpA or the SlyD orthologue of Pasteurella multocida may be partof the first fusion protein. The first and second fusion proteins areadded, simultaneously or consecutively, to a sample under investigationfor a specific antibody analyte. The antibody when present in the samplebinds to the target polypeptide units of the first and the second fusionprotein thereby bridging the target polypeptide parts of said first andsaid second fusion proteins resulting in an immunoreaction product orimmunocomplex.

Before, after or concomitant with the formation of an immunocomplex, asolid phase like, e.g., microbeads or an ELISA plate is added so thatthe first fusion protein binds to the solid phase. As a consequence thewhole immunoreaction product (i.e., the immunocomplex) comprising saidfirst fusion protein, the antibody to be detected and said second fusionprotein binds to the solid phase. After separation of the solid phasefrom the liquid phase the presence of the immunoreaction product can bedetected. As an alternative, the chaperone units present in the firstfusion protein may be used as chaperone units for the second fusionprotein and vice versa. However, the chaperone units should preferablybe different in both fusion proteins because of possible (unpredictable)non-analyte specific cross-linking of the fusion proteins due to thepresence of antibodies against these chaperones in the sample. As analternative, a highly specific DAGS immunoassay would also be feasiblewith identical chaperone fusion partners on either side of the assay. Inthis scenario, the developer of the assay must take into account ashighly probable that antibodies against the used fusion partner arepresent in a substantial fraction of human sera. These antibodies wouldbridge the signaling polypeptide to the solid phase, raise the signaland thus evoke falsely positive results. In order to avoid suchinterferences, the fusion partner (i.e., the chaperone unit) would beadded to the sample in a highly polymerized and unlabeled form as ananti-interference substance. The anti-interference substance is designedto efficiently capture immunoglobulins directed against the fusionpartner, the linker segments, the spacer and tag sequences and all othermoieties which are not part of the genuine antigen. By virtue of itshigh epitope density, a chemically polymerized (i.e., cross-linked)anti-interference substance is able to efficiently compete with thelabeled fusion polypeptide for the binding of anti-chaperone antibodies.This way, interferences due to immunoglobulins with unwantedspecificities can be ruled out in a convenient and reliable fashion. Asa sample all biological liquids like body fluids may be used.Preferably, blood, serum, plasma, urine or saliva are used.

The labeling or signaling group can be selected from any knowndetectable marker groups, such as dyes, luminescent labeling groups suchas chemiluminescent groups, e.g., acridinium esters or dioxetanes, orfluorescent dyes, e.g., fluorescein, coumarin, rhodamine, oxazine,resorufin, cyanine and derivatives thereof. Other examples of labelinggroups are luminescent metal complexes, such as ruthenium or europiumcomplexes, enzymes, e.g., as used for ELISA or radioisotopes.

The attachment of the immunocomplex or immunoreaction product to thesolid phase may be carried out using one partner of a bioaffinitybinding pair like, e.g., biotin and streptavidin. Preferably, biotin iscoupled to the fusion protein according to the invention. Thisbiotin-fusion protein-conjugate binds with high affinity to astreptavidin coated solid phase.

Examples of analytes are all pathogens and antibodies against thesepathogens mentioned under the “target polypeptide” section. For example,according to the invention preferably antibodies against HIV (humanimmunodeficiency virus), HTLV-1/HTLV-II (human T-cell lymphotropic virusI & II), HCV (hepatitis C virus), HBV (hepatitis B virus), HAV(hepatitis A virus), HCMV (human cytomegalic virus), HSV-1/-2 (herpessimplex virus 1 & 2), EBV (Epstein-Barr virus), varicella zoster virus,human herpesvirus 6, human herpesvirus 7, human herpesvirus 8, rubellavirus, Treponema pallidum, Helicobacter pylori, Borrelia (burgdorferi,afzeli, garinii). Trypanosoma cruzi, and Toxoplasma gondii canspecifically be detected.

Yet another embodiment of the invention is a reagent kit for thedetection of antibodies against an analyte, containing a fusion proteincomprising at least one polypeptide sequence corresponding to SlpA andat least one polypeptide sequence corresponding to a target peptide.Further parts of such a reagent kit are known to someone skilled in theart and include buffers, preservatives, labeling substances andinstructions for use.

Further embodiments of the invention include the use of a recombinantlyor synthetically produced fusion protein according to the invention as ameans for the reduction of interferences in an immunoassay and its usefor immunization of laboratory animals and the production of a vaccine.

Another embodiment of the invention relates to a composition comprisinga recombinantly or synthetically produced fusion protein comprising atleast one polypeptide sequence corresponding to SlpA and at least onepolypeptide sequence corresponding to a target peptide and apharmaceutically acceptable excipient.

According to the invention SlpA can be used as a folding helper fortarget polypeptides by adding SlpA in purified form to a targetpolypeptide, which includes addition of SlpA to any protein formulationas a stabilizing or solubilizing agent. For example, SlpA and relatedfolding helpers from the FKBP family of peptidyl-prolyl-cis/transisomerases can be added during or after the process of biotechnologicalproduction of target polypeptides thereby conferring solubility orthermal stability to the target polypeptide. Such biotechnologicalapplications include for example large-scale industrial production ofenzymes, peptide hormones such as, e.g., insulin, or, more generally,proteins of commercial interest.

In a further embodiment of the invention SlpA can be used as an additivein immunoassays to reduce or suppress immunological cross-reactions orinterferences that evoke erroneously positive results, particularly in adouble antigen sandwich immunoassay format.

More specifically, in an immunoassay SlpA-X or SlpA-SlpA-X fusionproteins may be used as antigens for the detection of an immunoglobulinanalyte wherein X is the target polypeptide to which theanalyte-specific antibodies bind. To reduce interferences, SlpA orSlpA-SlpA would be added as an anti-interference substance to avoidimmunological cross-reactions via the chaperone unit. Preferably, SlpAor SlpA-SlpA would be added in a chemically polymerized form in order toincrease the epitope density and to foster the binding of IgG and IgMmolecules directed to SlpA, the linker segments or the hexa-histidinetag (SEQ ID NO: 18).

SlpA confers solubility and stability on target molecules, but it might,as any other moiety or group different from the very target molecule,evoke immunological cross-reactions that compromise the specificity ofthe respective immunoassay. In order to overcome this specificityproblem, an unlabeled variant of SlpA or SlpA-SlpA is implemented in theimmunoassay reagents in a polymerized form. This SlpA or SlpA-SlpApolypeptide comprises all of the elements that might evokecross-reactions such as the SlpA unit itself, any linker or spacersegments, the hexa-histidine (SEQ ID NO: 18) or other tag motifs andeven the label moieties, albeit in an inactivated form. Due to chemicalcross-linking, these potential interference-prone motifs are presentedto the cross-reacting antibodies in a high epitope density, which iswell-suited to bind to and thus neutralize these potentially interferingantibodies. Besides this anti-interference effect, the SlpA or SlpA-SlpApolymer may even have additional advantageous effects: as a highlypolymeric chaperone it should be able to adsorb to hydrophobic surfaceareas of any solid surface (such as beads, microtiter plates and tube orvessel walls) and thus reduce unspecific adsorption of the essentialimmunological components. Further, it may contribute to the solubilityof other immunological components by virtue of its chaperone features,which might even be more pronounced in its polymerized form.

The examples that follow illustrate the invention further.

Example 1 Cloning and Purification of SlpA and SlyD Fusion Polypeptides

Cloning of Expression Cassettes

On the basis of the pET24a expression plasmid of Novagen (Madison, Wis.,USA) an expression cassette encoding the SlyD or SlpA fusionpolypeptides was obtained. The sequence of the gp41 ectodomain wasretrieved from the SwissProt database. A synthetic gene encoding gp41(aa 536-681) with a glycine-rich linker region fused in frame to theN-terminus was purchased from Medigenomix (Martinsried, Germany). BamHIand XhoI restriction sites were at the 5′ and the 3′ ends of the codingregion, respectively. A further synthetic gene encoding two SlpA units(residues 1-146 and 2-149 according to SEQ ID NO: 1, SwissProt accessionno. P0AEM0) connected via a glycine-rich linker region and encompassingpart of a further linker region at the C-terminus were likewisepurchased from Medigenomix. NdeI and BamHI restriction sites were at the5′ and 3′ ends of this cassette, respectively. The genes and therestriction sites were designed to enable the in frame fusion ofSlpA-SlpA and the gp41 ectodomain fragment by simple ligation. In orderto avoid inadvertent recombination processes and to increase the geneticstability of the expression cassette in the E. coli host, the nucleotidesequences encoding the SlpA units were degenerated as well as thenucleotide sequences encoding the extended linker regions. i.e.,different codon combinations were used to encode identical amino acidsequences.

The pET24a vector was digested with NdeI and XhoI and the cassettecomprising tandem-SlpA fused in frame to the HIV-1 gp41 ectodomainfragment 536-681 was inserted. Expression cassettes comprising SlyD ortandem SlyD instead of SlpA or tandem SlpA were constructed accordingly,as well as expression cassettes comprising target polypeptides differentfrom gp41. All recombinant fusion polypeptide variants contained aC-terminal hexahistidine tag (SEQ ID NO: 18) to facilitateNi-NTA-assisted purification and refolding. QuikChange (Stratagene, LaJolla, Calif., USA) and standard PCR techniques were used to generatepoint mutations, deletion and extension variants or restriction sites inthe respective expression cassettes.

The drawing below shows a scheme of the resulting HIV-1 gp41 ectodomainfragment 536-681 bearing two tandem SlpA chaperones fused in frame toits N-terminal end.

The insert of the resulting plasmid was sequenced and found to encodethe desired fusion protein. The complete amino acid sequence is shown inSEQ ID NO: 4. The amino acid sequence of the linker L is shown is SEQ IDNO: 17.

Purification of SlpA, SlyD and Fusion Proteins Comprising SlpA, SlyD,and FkpA

SlyD, SlpA, and all fusion protein variants were purified by usingvirtually identical protocols. E. coli BL21 (DE3) cells harboring theparticular pET24a expression plasmid were grown at 37° C. in LB mediumplus kanamycin (30 μg/ml) to an OD₆₀₀ of 1.5, and cytosolic overexpression was induced by adding 1 mM isopropyl-β-D-thiogalactoside.Three hours after induction, cells were harvested by centrifugation (20min at 5000 g), frozen and stored at −20° C. For cell lysis, the frozenpellet was resuspended in chilled 50 mM potassium phosphate pH 8.0, 7.0M GdmCl, 5 mM imidazole and the suspension was stirred for 2 h on ice tocomplete cell lysis. After centrifugation and filtration (cellulosenitrate membrane, 0.45 μm/0.2 μm), the lysate was applied onto a Ni-NTAcolumn equilibrated with the lysis buffer including 5.0 mM TCEP. Thesubsequent washing step was tailored for the respective target proteinand ranged from 5-15 mM imidazole in 50 mM potassium phosphate pH 8.0,7.0 M GdmCl, 5.0 mM TCEP. At least 10-15 volumes of the washing bufferwere applied. Then, the GdmCl solution was replaced by 50 mM potassiumphosphate pH 7.8, 100 mM KCl, 10 mM imidazole, 5.0 mM TCEP to induceconformational refolding of the matrix-bound protein. In order to avoidreactivation of copurifying proteases, a protease inhibitor cocktail(Complete EDTA-free, Roche) was included in the refolding buffer. Atotal of 15-20 column volumes of refolding buffer were applied in anovernight reaction. Then, both TCEP and the Complete EDTA-free inhibitorcocktail were removed by washing with 3-5 column volumes 50 mM potassiumphosphate pH 7.8, 100 mM KCl, 10 mM imidazole. The native protein wasthen eluted by 250 mM imidazole in the same buffer. Protein-containingfractions were assessed for purity by Tricine-SDS-PAGE and pooled.Finally, the proteins were subjected to size-exclusion-chromatography(SUPERDEX HiLoad, GE Healthcare Bio-Sciences AB) and theprotein-containing fractions were pooled and concentrated in an Amiconcell (YM10).

After the coupled purification and refolding protocol, yields of roughly5-20 mg could be obtained from 1 g of E. coli wet cells, depending onthe respective target protein.

Example 2 Spectroscopic Measurements

Circular dichroism spectroscopy (CD) is the method of choice to assessboth the secondary and the tertiary structure in proteins. Ellipticityin the aromatic region (260-320 nm) reports on tertiary contacts withina protein (i.e., the globular structure of a regularly folded protein),whereas ellipticity in the amide region (190-250 nm) reflects regularrepetitive elements in the protein backbone, i.e., the secondarystructure.

Protein concentration measurements were performed with an Uvikon XLdouble-beam spectrophotometer. The molar extinction coefficients (ε₂₈₀)were determined by using the procedure described by Pace (1995), ProteinSci. 4, 2411-2423.

Near-UV CD spectra were recorded with a Jasco-720 spectropolarimeterwith a thermostated cell holder and converted to mean residueellipticity. The buffer was 50-150 mM potassium phosphate pH 7.5, 100 mMKCl, 1 mM EDTA. The path length was 0.5 cm or 1.0 cm, the proteinconcentration was 20-500 μM. The band width was 2 nm, the scanning speedwas 50 nm/min at a resolution of 0.5 nm and the response was 1 or 2 s.In order to improve the signal-to-noise ratio, spectra were measurednine times and averaged.

Far-UV CD spectra were recorded with a Jasco-720 spectropolarimeter witha thermostated cell holder and converted to mean residue ellipticity.The buffer was 10 mM potassium phosphate pH 7.5, 25 mM KCl, 0.5 mM EDTA.The path length was 0.2 cm, the protein concentration ranged between 2.5and 20 μM. The band width was 2 nm, the scanning speed was 50 nm/min ata resolution of 0.5 nm and the response was 1 or 2 s. In order toimprove the signal-to-noise ratio, spectra were measured nine times andaveraged.

Example 3 Coupling of Biotin and Ruthenium Moieties to the FusionProteins

The lysine ε-amino groups of the fusion polypeptides were modified atprotein concentrations of 10-20 mg/ml with N-hydroxy-succinimideactivated biotin and ruthenium labels, respectively. The label/proteinratio varied from 2:1 to 5:1 (mol:mol), depending on the respectivefusion protein. The reaction buffer was 150 mM potassium phosphate pH8.0, 100 mM KCl, 1 mM EDTA. The reaction was carried out at roomtemperature for 15 min and stopped by adding buffered L-lysine to afinal concentration of 10 mM.

To avoid hydrolytic inactivation of the labels, the respective stocksolutions were prepared in dried DMSO (seccosolv quality, Merck,Germany). DMSO concentrations up to 15% in the reaction buffer were welltolerated by all fusion proteins studied. After the coupling reaction,unreacted free label was removed by passing the crude protein conjugateover a gel filtration column (SUPERDEX 200 HiLoad).

Example 4 Immunological Reactivity of the Polypeptide Fusion Proteins

The immunological reactivity (i.e., the antigenicity) of the differentfusion proteins was assessed in an automated ELECSYS 2010 analyzer(Roche Diagnostics GmhH). Measurements were carried out in the doubleantigen sandwich format.

Signal detection in ELECSYS 2010 is based on electrochemiluminescence.The biotin-conjugate (i.e., the capture-antigen) is immobilized on thesurface of a streptavidin coated magnetic bead whereas thedetection-antigen bears a complexed Ruthenium cation (switching betweenthe redox states 2+ and 3+) as the signaling moiety. In the presence ofa specific immunoglobulin analyte, the chromogenic ruthenium complex isbridged to the solid phase and emits light at 620 nm after excitation ata platinum electrode. The signal output is in arbitrary light units.

Fusion polypeptides containing HSV-1 antigen gG1 (amino acids 26-189,see SEQ ID NOs: 7 and 8) as HSV-1 specific antigenic sequence were usedin the assay for both the capture and the detection antigen. The gG1antigen was either fused to SlpA or SlyD. In the double antigen sandwichimmunoassay, a SlpA-gG1 (26-189)-biotin conjugate was applied togetherwith a SlpA-gG1 (26-189)-ruthenium complex conjugate (invention) at aconcentration of 100 ng/ml each. As well, a SlyD-gG1 (26-189)-biotinconjugate was applied together with a SlyD-gG1 (26-189)-rutheniumcomplex conjugate (comparison) at a concentration of 100 ng/ml each.

The biotin and the ruthenium conjugates of the fusion polypeptidevariants of gG1 (26-189) were assessed for their reactivity againstanti-HSV-1 positive sera at concentrations of 100 ng/ml each. In allmeasurements, unlabeled chemically polymerized SlyD-SlyD was implementedin the reaction buffer as an anti-interference substance to avoidimmunological cross reactions via the chaperone fusion unit. Elevenanti-HSV-1 negative human sera were used as controls.

To ascertain the thermo tolerance of the fusion proteins, SlyD-gG1 andSlpA-gG1 were subjected to harsh temperature conditions as follows:SlyD-gG1 and SlpA-gG1 (proteins in 50 mM potassium phosphate pH 7.5, 100mM KCl, 1 mM EDTA) were incubated overnight at 60° C. The concentrationof the gG1-biotin conjugates was roughly 1.3 mg/ml each, theconcentration of the gG1-ruthenium conjugates was roughly 0.6 mg/mleach. Subsequently, the thermally stressed samples were assessed fortheir residual immunological reactivity in an ELECSYS 2010 automatedanalyzer under the experimental conditions described above. Unchallengedsamples (stored at 2-8° C.) of SlyD-gG1 and SlpA-gG1 were used as areference.

The outcome of the experiments is shown in Table 1 (FIG. 9).

Table 1 depicts the immunological reactivity of SlpA-gG1(26-189) andSlyD-gG1(26-189) with human anti-HSV-1 positive and anti-HSV-1 negativesera in an automated ELECSYS analyzer as described in Example 4. Shownis the performance of both antigen variants before and after a harshovernight heat-treatment at 60° C. The outcome of the experimentsclearly demonstrates the superiority of heat-stressed SlpA-gG1(26-189)over heat-stressed SlyD-gG1 (26-189) in a twofold manner. Firstly, thespecific signal recovery with anti-HSV-1 positive sera (upper half ofTable 1) is significantly higher with the heat-challenged SlpA fusionpolypeptide. Secondly, the increase in unspecific background signal withanti-HSV-1 negative sera (lower half of Table 1) is significantly lowerwith the heat-challenged SlpA fusion polypeptide. We observe aconsiderable increase in the background signal after heat-treatment ofthe SlyD fusion polypeptides (see right column, about 100 to 900%increase in the background signal).

When using SlpA fusion polypeptides according to the invention, however,the increase in background signal after heat stress is negligibly low,i.e., it is below 20% in all but one cases. In that one case, (serumsample Trina 07/06-533) there is an increase in background signal of48%. The very same sample (Trina 07/06-533) shows an increase inbackground signal of more than 800% when the SlyD fusion polypeptide isused instead. This shows that even with difficult samples thatinherently evoke slightly elevated background signals SlpA fusionpolypeptides can substantially reduce the background signal. Lowbackground signals are highly desired in the development of immunoassaysbecause they enable the manufacturer to set a low cut-off value.Generally, reduced background signals are required for an increasedassay performance with respect to sensitivity. The reason is thatsamples yielding a signal above the cut-off value are considered aspositive (i.e., the samples are assumed to contain the analyte understudy); samples yielding a signal below the cut-off value are consideredas negative. It is therefore easy to understand why a low cut-off isutterly needed: the lower the cut-off is, the higher is the probabilitythat samples that contain low analyte concentrations (and concomitantlyyield low signals) will be correctly found as low positive. Thus, thesensitivity of an immunoassay may be increased by lowering thebackground signal that inherently originates from its immunologicalcomponents. The use of SlpA as a folding helper thus clearly contributesto improve and warrant the long-term sensitivity of an immunoassay.

To sum up, fusion polypeptides containing SlpA increase both thestability and the solubility of the fused target polypeptides, inparticular under critical conditions (such as thermal stress), whichusually would compromise the native fold and lead to aggregationprocesses. In brief, SlpA is an excellent folding helper which protectsthe integrity of its client proteins even under very unfavorableconditions, facilitates their refolding into an active conformation andkeeps them in solution. Fusion to SlpA or, more simply, addition of SlpAis therefore an excellent means to stabilize target molecules in proteinformulations intended for diagnostic and other biotechnologicalpurposes.

1. A recombinant fusion protein comprising an E. coli SlpA chaperonepolypeptide sequence and a target polypeptide, wherein the targetpolypeptide comprises a herpes simplex virus 1 (HSV-1) antigen gG1. 2.The recombinant fusion protein of claim 1, wherein the SlpA chaperonepolypeptide sequence comprises SEQ ID NO:
 2. 3. The recombinant fusionprotein of claim 1, wherein the SlpA chaperone polypeptide sequencecomprises amino acids 1-147 of SEQ ID NO:
 2. 4. The recombinant fusionprotein of claim 1 comprising SEQ ID NO:
 7. 5. The recombinant fusionprotein of claim 1 comprising amino acids 1-337 of SEQ ID NO:
 7. 6. Acomposition comprising a recombinant fusion protein according to claim 1and a pharmaceutically acceptable excipient.
 7. A recombinant fusionprotein comprising an E. coli SlpA chaperone polypeptide sequence and atarget polypeptide, wherein the target polypeptide comprises at leastone of a herpes simplex virus 1 (HSV-1) antigen gG1 and a herpes simplexvirus 2 (HSV-2) antigen gG2.
 8. The recombinant fusion protein of claim7, wherein the SlpA chaperone polypeptide sequence comprises SEQ ID NO:2.
 9. The recombinant fusion protein of claim 7, wherein the SlpAchaperone polypeptide sequence comprises amino acids 1-147 of SEQ ID NO:2.
 10. A composition comprising a recombinant fusion protein accordingto claim 7 and a pharmaceutically acceptable excipient.